Decoding links between the RNA polymerase II C-terminal domain and histone modifications

RNA聚合酶II(RNAPII)延伸复合体是真核生物基因表达调控途径的关键枢纽。除了其固有的mRNA合成功能外，RNAPII延伸复合体还调控共转录的mRNA处理和输出、共价组蛋白修饰和染色质结构。协调所有这些功能的分子平台是RNAPII大亚基的C末端结构域(CTD)。这个真核生物特有的结构域由一个保守的七肽基序(Y1S2P3T4S5P6S7)的多个重复组成。人们普遍认为，在给定的RNAPII中，CTD重复残基的磷酸化模式构成了一个“代码”，它决定了它所涉及的因素的星座。鉴于CTD的复杂性质，各因素如何直接识别CTD在很大程度上还不清楚。 这项提议旨在破译组蛋白修饰酶识别CTD的基础。延长的RNAPII在转录的基因上产生高度刻板的组蛋白修饰模式，与特定的CTD磷酸化形式相关。CTD和染色质修饰模式在体内如何协调，以及RNAPII延长复合体与相关酶之间相互作用的性质，是重要的悬而未决的问题。我们将通过以下具体目标解决这些问题： 1)在模式真核生物S.pombe中，使用合成肽文库和一组CTD突变体，定义了用于选择染色质修饰复合体的CTD“代码”。这将允许对调节纯化的天然复合体与CTD重复关联的磷酸化模式进行全面评估。我们还将对染色质修饰剂和磷酸化CTD多肽之间的络合物进行生化和结构表征。 2)研究调控转录基因中组蛋白甲基化的CTD修饰途径的实现和功能。 除了破译转录和染色质修饰之间的联系外，我们预计我们的方法将产生关于因素如何参与RNAPII延长复合体的一般见解。

The RNA polymerase II (RNAPII) elongation complex is a key hub for regulatory pathways controlling gene expression in eukaryotes. Aside from its intrinsic mRNA synthesis function, the RNAPII elongation complex governs co-transcriptional mRNA processing and export, covalent histone modifications, and chromatin structure. The molecular platform coordinating all of these functions is the C-terminal domain (CTD) of the RNAPII large subunit. This eukaryote-specific domain consists of multiple repeats of a conserved heptad motif (Y1S2P3T4S5P6S7). It is widely presumed that patterns of phosphorylation of CTD repeat residues within a given RNAPII constitute a “code” which dictates the constellation of factors with which it is engaged. Given the complex nature of the CTD, how factors directly recognize the CTD is, for the most part, poorly understood. This proposal aims to decipher the basis for CTD recognition by histone-modifying enzymes. Elongating RNAPII generates a highly stereotyped pattern of histone modifications at transcribed genes that correlates with specific phosphorylated forms of the CTD. How the CTD and chromatin modification patterns are coordinated in vivo, as well as the nature of the interactions between the RNAPII elongation complex and the relevant enzymes, are important unresolved issues. We will address these questions through the following specific aims: 1) Definition of the CTD “code” for select chromatin-modifying complexes using a library of synthetic peptides and a set of CTD mutants in the model eukaryote S. pombe. This will allow comprehensive assessment of the pattern of phosphorylations that regulate association of purified native complexes with CTD repeats. We will also carry out biochemical and structural characterization of complexes between chromatin modifiers and phosphorylated CTD peptides. 2) Investigation of the implementation and function of a CTD modification pathway regulating histone methylation in transcribed genes. In addition to deciphering the links between transcription and chromatin modification, we expect that our approach will yield general insights into how factors engage the RNAPII elongation complex.